Abstract

Cytokine production in the tumor microenvironment is a key regulatory mechanism for tumor progression. We reported that IL-10 is produced in more than 85% of freshly isolated metastasic melanoma cell suspensions. That IL-10 production decreases when melanoma cells are maintained in vitro suggested that an interaction between melanoma cells and the tumor microenvironment is important for IL-10 production by melanoma cells.

We have demonstrated that IL-10 production by both fresh and established metastatic melanoma cells is increased in response to recombinant human IL-6 (rhIL-6) (Invitrogen). The stimulative effect of rhIL-6 is completely blocked by anti-IL-6 antibody (BD bioscience) and anti-IL-6 receptor antibody (BD bioscience). We concluded that a pro-inflammatory cytokine, IL-6 has a key role on the induction of IL-10 production by melanoma cells. In this study, we investigated the signal pathways that transduce IL-6 receptor activation into IL-10 production in a metastatic human melanoma cell line (JB) using pharmacological inhibitors or siRNAs for various kinases. Inhibitors were added to quiescent JB cells, 1 hour prior to a submaximum dose of IL-6 (2 ng/ml). After 24 hour stimulation, aliquots of culture media were assayed for IL-10 by ELISA. It was found that a JAK inhibitor, JAK inh1, suppressed 90% of IL-6-induced IL-10 production by JB cells compare to control. In parallel, cells were fixed with TCA, and the total proteins were subjected to phospho-immunoblotting assay. IL6 stimulation induced a 96-fold increase in STAT3 phosphorylation within 30 min, and the phosphorylation was sustained for 24 hours leading IL-10 production. JAK inhibitor-1 eliminated IL-10 production and STAT3 phosphorylation, suggesting a dominant role of JAK/STAT3 in IL-6-induced signals. ERK1/2 phosphorylation was spontaneously high in quiescent JB cells, and elevated 37% in response to IL-6 stimulation. Inhibition of ERK phosphorylation with inhibitors for c-Raf (GW5074) and MEK (U0126) eliminated IL-6-induced IL-10 production. A PI3K inhibitor (LY294002) suppressed IL-10 production without the inhibition of STAT3 or ERK phosphorylation. Rapamycin, an mTOR inhibitor, showed only a marginal effect on IL-10 production, indicating a minimum contribution of mTOR to the PI3K-mediated IL-10 production in melanoma. Thus, the spontaneous ERK phosphorylation and PI3K activity are necessary for STAT3-mediated IL-10 production in melanoma. IL-10 is purported to surpress anti-tumor cellular immune responses. Therefore, inhibitors that block IL-6-induced IL-10 production are expected to potentiate an anti-tumor immune response at tumor sites.